Metabolic Engineering Of Corynebacterium Glutamicum For L-valine Production

L-valine, one of the branched chain amino acids (BCAAs), is an essential nutrient withvariety of functions and widely used as component of food, cosmetics, pharmaceuticals andfeed ingredient, with significant increase of market demand. Industrial fermentation is themajor method to produce L-valine, and Corynebacterium glutamicum is one of most usedbacteria. L-valine producing strains of C. glutamicum were usually obtained via isolation andmultiple random mutations, therefore, they have undefined genetic background andnegative phenotype. Recently, metabolic engineering showed superior potential for L-valineproduction in C. glutamicum. In this study, Lrp regulation on L-valine metabolism wasinvestigated in C. glutamicum wild type ATCC13869and L-valine producing strain VWB-1;then metabolic engineering was used to increase L-valine production in both strains. Themain results are listed below:(1) The global regulator protein Lrp could regulate the expression of several genesrelated to L-valine metabolism in various bacteria, while its regulation of L-valine remainsunclear in C. glutamicum. Lrp from ATCC13869and Lrp1from VWB-1were overexpressedin both of them, resulting L-valine production increased significantly. Lrp1worked betterthan Lrp.(2) RT-PCR analysis showed that Lrp and Lrp1could increasing the transcriptionallevels of genes related to L-valine biosynthesis (ilvA, ilvBN, ilvC, ilvD) and export (brnFE),which might be the reason for the improved L-valine production. Further study suggestedthat a single amino acid substitution (Arg39Trp) in Lrp1might affect Lrp binding to DNA,leading to higher L-valine production, especially incontaning mutations in the region LF1between lrp and brnF.(3) Metabolic engineering was carried out in VWB-1. Deletion of genes pgi and brnQled no improve to L-valine production, while deletion of gene ldh and fermentation underanoxic conditions increased L-valine yield27.8%to35.9g/L. Furthermore, by co-expressingLrp1and BrnFE, L-valine production of flask and5L fed-batch fermentation reached38.1and42.1g/L, with35.6%and48.9%increased respectively.(4) L-valine producing strain was developed from C. glutamicum ATCC13869throughdeletion of three genes aceE, alaT and ilvA and overexpression of six genes lrp1、brnF、brnE、ilvB、ilvN and ilvC. The final strain WCC003/pJYW-4-ilvBNC1-lrp1-brnFE couldproduce23.9and36.6g/L L-valine in flask cultivation and5L fed-batch fermentation.(5) The optimal growth conditions for L-valine production in WCC003/pJYW-4-ilvBNC1-lrp1-brnFE are using the second-grade seeds, adding0.4g/L isoleucine,3x10g/Lpotassium acetate (0,24and48h) and2mM sodium pyruvate. Then the strain couldproduce28.5g/L L-valine in flask cultivation and51.2g/L L-valine in5L fed-batchfermentation after96h, without detectable by-product amino acids such as L-alanine and L-isoleucine.